CN104568752A - Centrifugal force based parallel high-throughput single-molecule force spectrum testing method - Google Patents
Centrifugal force based parallel high-throughput single-molecule force spectrum testing method Download PDFInfo
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- CN104568752A CN104568752A CN201410819837.9A CN201410819837A CN104568752A CN 104568752 A CN104568752 A CN 104568752A CN 201410819837 A CN201410819837 A CN 201410819837A CN 104568752 A CN104568752 A CN 104568752A
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Abstract
The invention discloses a centrifugal force based parallel high-throughput single-molecule force spectrum testing method. The testing method comprises the following steps: 1) fixing an optical imaging system on a rotating platform, wherein the optical imaging system comprises a camera, an optical microscope and an LED lamp; 2) connecting microspheres with one end of the single molecule, and connecting the other end of the molecule with the surface of a molecular substrate which can be provided with another molecule connected with the molecule; 3) placing the molecular substrate with the molecule and the microspheres on the rotating platform, integrating the imaging system, the molecular substrate and the rotating platform to form a whole body, controlling the revolving speed of the rotating platform to control the centrifugal force subjected to the microspheres; 4) enabling the optical microscope and the camera to trace and record the microsphere positions, so as to obtain the molecular deformation information and the molecular interaction information. The centrifugal force based parallel high-throughput single-molecule force spectrum testing method disclosed by the invention can be used for applying the centrifugal force to thousands of microspheres, meanwhile tracing and measuring the positions of the microspheres, and further simultaneously performing parallel measurement to the plurality of molecular force spectrums.
Description
Technical field
The present invention relates to single molecule force spectroscopy fields of measurement, in particular, relate to a kind of parallel high flux single molecule force spectroscopy method of testing based on centrifugal force.
Background technology
Single molecule force spectroscopy device mainly comprises: light tweezer, magnetic tweezer and atomic force microscope etc.Light tweezer utilizes light probe to carry out manipulation to unimolecule and ergometry spectrum; Magnetic tweezer is by applying magnetic field to the monomolecular magnetic bead of link, producing acting force thus handle unimolecule magnetic bead; Atomic force microscope sticks on measuring probe by unimolecule, measured single molecule force spectroscopy by probe deformation.These three kinds of devices can produce a constant force to individual molecule, and this molecule can stretch deformation thereupon.But the magnetic tweezer of light tweezer, atomic force microscope and routine usually can only one-shot measurement molecule, cannot obtain the experimental result with statistical significance.Special magnetic tweezer can measure multiple unimolecule simultaneously, but it is very difficult to the unitarity applying magnetic force, and the scope of its power applied is less, has significant limitation.
Summary of the invention
The object of the invention is to overcome deficiency of the prior art, a kind of parallel large flux single molecule force spectroscopy method based on centrifugal force is provided, centrifugal force can be applied to thousands of microballoons simultaneously, and simultaneously tracking measurement microballoon position, and then carry out parallel measurement to multiple molecular force spectrum, and the scope applying centrifugal force in the method is can from skin ox level to receiving ox level simultaneously.
The object of the invention is to be achieved through the following technical solutions:
Based on the parallel high flux single molecule force spectroscopy method of testing of centrifugal force, comprise the following steps:
1) be fixed on rotation platform by optical imaging system, described optical imaging system is made up of camera, optical microscope and LED;
2) microballoon is connected to unimolecule one end, the molecule other end is connected to molecule substrate surface, described molecule substrate also can be provided with the another kind of molecule be connected with described molecule;
3) be placed on rotation platform by the molecule substrate being provided with molecule and microballoon, described imaging system and molecule substrate and rotation platform form one, are controlled the size of centrifugal force suffered by described microballoon by the rotating speed controlling rotation platform;
4) use optical microscope and camera tracing record microballoon position, thus obtain the deformation data of molecule and intermolecular interaction information.
By changing rotation platform rotating speed, the molecule be connected on microballoon is applied to the drawing force of different size.
Under described optical microscope, the diffraction stripe of described microballoon changes with microballoon and object lens distance and changes, record the Changing Pattern of described microballoon diffraction stripe and object lens distance, obtain microballoon and be subject to the change of the distance before and after centrifugal force and between object lens, thus obtain described molecule and be subject to interactional change between the deformation characteristics after centrifugal force or two quasi-molecules.
Use out of focus imaging technique to carry out three-dimensional position tracking to microballoon, obtain microballoon positional information in three dimensions, thus obtain described molecule and be subject to interactional change between the deformation characteristics after centrifugal force or two quasi-molecules.
Described molecule is the DNA sample prepared by PCR.
Described microballoon is made up of polystyrene microsphere, silicon ball or magnetic bead wherein one or more.
Described microsphere diameter is 1 μm-25 μm.
Described molecule substrate is slide.
The range of speeds of described rotation platform is 0-700rpm.
The acting force scope applied unimolecule in the method from 0.1 skin ox level to receiving ox level, and is applicable to the measurement of multiple single molecule force spectroscopy.
Compared with prior art, the beneficial effect that technical scheme of the present invention is brought is:
1, the molecule substrate in the present invention can be arranged a lot of the microballoons be connected with molecular sample simultaneously, and apply centrifugal force to microballoon by rotation platform simultaneously, the centrifugal force produced microballoon is identical, has very high homogeneity.
2, the optical imaging system energy real-time follow-up microballoon position in the present invention record, therefore can concurrent testing single molecule force spectroscopy fast and accurately.
3, in the present invention rotation platform maximum speed can reach 700rpm can from skin ox level to receiving ox level to the acting force scope that molecule applies.
4, the inventive method is applicable to the measurement of multiple single molecule force spectroscopy.
Embodiment
The present invention proposes a kind of parallel high flux single molecule force spectroscopy method based on centrifugal force, comprises the following steps:
Step 1: build optical imaging system, and be fixed on High Rotation Speed platform, optical imaging system is by Ethernet camera, and optical microscope and high energy red LED lamp form;
Step 2: microballoon is connected to molecule one end, is connected to molecule substrate surface by the other end of molecule, or connects another kind of molecule at molecule substrate surface, and two quasi-molecules are by the connection that specifically interacts;
Step 3: the molecule substrate including microballoon and molecule is fixed on High Rotation Speed platform, now optical imaging system, molecule substrate and High Rotation Speed platform composition one, rotate microballoon applying centrifugal force by controlling turntable, and control the size of centrifugal force suffered by microballoon by controlling rotating speed;
Step 4: use optical microscope and camera tracing record microballoon position, thus obtain molecular deformation and interaction information.
First the present invention controls High Rotation Speed platform and rotates, and reaches a constant rotational speed at short notice, makes the molecule be connected on microballoon be subject to a constant tensile force.
Further, under optical microscope, the diffraction stripe of microballoon changes with the object lens distance of microballoon and optical microscope and changes, the displacement of microballoon in microscopes optical axis direction can be judged by the situation of change of the shape of microballoon diffraction spot, the Changing Pattern of record microballoon diffraction stripe and microballoon object lens distance, obtain microballoon and be subject to the change of the distance before and after centrifugal force and between object lens, thus obtain the molecule be connected between microballoon and substrate and be subject to the deformation after centrifugal force, or the interactional change between two quasi-molecules.
By using the camera interval setting-up time imaging that is connected on microscope, obtain the interaction distance between the deformation process be subject under specific external force in time and two quasi-molecules of multiple molecule in imaging region over time.
According to present pre-ferred embodiments, described molecule is the DNA sample prepared by PCR (Polymerase ChainReaction, PCR).One end that the rear purifying acquisition of PCR reaction is about 3.5kb is digoxigenin labeled, the other end is biotin labeled double-stranded DNA.This DNA and finishing are had the slide of DigiTAb (molecule substrate) to react, make DNA be connected to surface of glass slide, the diameter that the other end and Streptavidin are modified is that the microballoon reacting phase of 5 microns is connected.Wherein microballoon is polystyrene microsphere, silicon ball or magnetic bead one or more formations wherein, and it is 1 μm-25 μm that diameter range can be.
Slide (molecule substrate) with molecular sample and microballoon is positioned on High Rotation Speed platform, controls it and rotate, make it with rotating speed 200rpm constant speed rotary, the acting force of about 28 skin oxen can be produced microballoon used.The rotation brachium of High Rotation Speed platform is 350 millimeters, and maximum speed is 700rpm, can produce 0.1 Pi Niu-5 receive the acting force of ox to microballoon.
Use camera and microscope with 15 frame recording images per second, tracing study is connected to the motion process of microballoon before and after rotating of single DNA.Under 50X object lens, about have 80 microballoons to be recorded, wherein about 40 are pulled away from surface of glass slide about 1.1 microns after rotation simultaneously.This measurement result is consistent with measurement results such as atomic force microscope, magnetic tweezer, light tweezers.
Claims (10)
1., based on the parallel high flux single molecule force spectroscopy method of testing of centrifugal force, it is characterized in that, the method comprises the following steps:
1) be fixed on rotation platform by optical imaging system, described optical imaging system is made up of camera, optical microscope and LED;
2) microballoon is connected to unimolecule one end, the molecule other end is connected to molecule substrate surface, described molecule substrate also can be provided with the another kind of molecule be connected with described molecule;
3) be fixed on rotation platform by the molecule substrate being provided with molecule and microballoon, described imaging system, molecule substrate and rotation platform form one, are controlled the size of centrifugal force suffered by described microballoon by the rotating speed controlling rotation platform;
4) use optical microscope and camera tracing record microballoon position, thus obtain the deformation data of molecule and intermolecular interaction information.
2. the parallel high flux single molecule force spectroscopy method of testing based on centrifugal force according to claim 1, is characterized in that, by changing rotation platform rotating speed, the molecule be connected on microballoon is applied to the drawing force of different size.
3. the parallel high flux single molecule force spectroscopy method of testing based on centrifugal force according to claim 1, it is characterized in that, under described optical microscope, the diffraction stripe of described microballoon changes with microballoon and object lens distance and changes, record the Changing Pattern of described microballoon diffraction stripe and object lens distance, obtain microballoon and be subject to the change of the distance before and after centrifugal force and between object lens, thus obtain described molecule and be subject to interactional change between the deformation characteristics after centrifugal force or two quasi-molecules.
4. the parallel high flux single molecule force spectroscopy method of testing based on centrifugal force according to claim 1, it is characterized in that, out of focus imaging technique is used to carry out three-dimensional position tracking to microballoon, obtain microballoon positional information in three dimensions, thus obtain described molecule and be subject to interactional change between the deformation characteristics after centrifugal force or two quasi-molecules.
5. the parallel high flux single molecule force spectroscopy method of testing based on centrifugal force according to claim 1, it is characterized in that, described molecule is the DNA sample prepared by PCR.
6. the parallel high flux single molecule force spectroscopy method of testing based on centrifugal force according to claim 1, is characterized in that, described microballoon is made up of polystyrene microsphere, silicon ball or magnetic bead wherein one or more.
7. the parallel high flux single molecule force spectroscopy method of testing based on centrifugal force according to claim 1 or 6, is characterized in that, described microsphere diameter be 1 μ m ?25 μm.
8. the parallel high flux single molecule force spectroscopy method of testing based on centrifugal force according to claim 1, it is characterized in that, described molecule substrate is slide.
9. the parallel high flux single molecule force spectroscopy method of testing based on centrifugal force according to claim 1, is characterized in that, the range of speeds of described rotation platform be 0 ?700rpm.
10. the parallel high flux single molecule force spectroscopy method of testing based on centrifugal force according to claim 1, is characterized in that, the acting force scope applied unimolecule in the method from 0.1 skin ox level to receiving ox level, and is applicable to the measurement of multiple single molecule force spectroscopy.
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| CN107727732A (en) * | 2017-11-16 | 2018-02-23 | 上海交通大学 | One kind is used for protein-interacting group single molecule force spectroscopy method |
| CN109557341A (en) * | 2018-11-22 | 2019-04-02 | 中国科学院上海应用物理研究所 | A kind of accurate high-throughput single molecule force spectroscopy method |
| EP3420352A4 (en) * | 2016-02-25 | 2020-01-08 | Children's Medical Center Corporation | Spinning apparatus for measurement of characteristics relating to molecules |
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| US20100137120A1 (en) * | 2008-12-02 | 2010-06-03 | President And Fellows Of Harvard College | Spinning Force Apparatus |
| CN102507724A (en) * | 2011-09-28 | 2012-06-20 | 上海交通大学 | Parallel large-flux monomolecular force spectrum method based on magnetic field force |
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| CN1766104A (en) * | 2005-09-01 | 2006-05-03 | 上海交通大学 | Method for atom force microscope inducing single molecule DNA positoning mutation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| EP3420352A4 (en) * | 2016-02-25 | 2020-01-08 | Children's Medical Center Corporation | Spinning apparatus for measurement of characteristics relating to molecules |
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| US11913872B2 (en) | 2016-02-25 | 2024-02-27 | President And Fellows Of Harvard College | Spinning apparatus for measurement of characteristics relating to molecules |
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| CN109557341A (en) * | 2018-11-22 | 2019-04-02 | 中国科学院上海应用物理研究所 | A kind of accurate high-throughput single molecule force spectroscopy method |
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